Project Summary: Severe rickettsial infections can be life-threatening with pre-antibiotic case-fatality rates reported as high as 65%-80% in some case series. Increased endothelial permeability is a major component of the pathophysiology of severe rickettsial diseases. However, the key mechanisms by which rickettsiae mediate severe diseases targeting endothelial cells (ECs) remains elusive. Our long term goal is to develop a novel therapeutic strategy that can effectively treat patients at even a late stage of infection to address the public health problems due to this disease. The objectives of this proposal are to understand the mechanisms mediating increased EC permeability by rickettsiae and to correct this disorder by counteracting the specific mechanism. The hypothesis to be tested in this proposal is that rickettsiae increase EC permeability via activating NLRP3 inflammasome. This hypothesis will be tested by accomplishment of two Specific Aims. Aim 1 is to determine the activation of NLRP3 inflammasome by rickettsiae in ECs. NLRP3-knock (KO) mice and NLRP3-specific short hairpin RNA (shRNA) will be employed to study the activation of NLRP3 inflammasome in R. australi-infected mouse brain microvascular ECs and R. conorii-infected human ECs. The cooperation of NLRC5 with NLRP3 inflammasome activation will be examined. Aim 2 is to determine the contribution of NLRP3 inflammasome to mediating EC hyperpermeability in severe rickettsioses. We will determine both the in vivo and in vitro role of NLRP3 inflammasome in mediating EC hyperpermeability. Casp-1 pharmaceutical inhibitors will be employed in R. conorii-infected C3H/HeN mice to amend the EC hyperpermeability in vivo. It is believed that accomplishment of the specific aims of this proposal may provide novel insights into therapeutic interventions targeting increased microvascular permeability.